Escherichia coli synthesize selenium-containing tRNA when grown anaerobically with 0.1 to 1.0 MuM Se-75 labeled selenite or selenate. The level of incorporation was relatively independent of medium sulfate concentration in the range 0.4 to 8.0 mM, implying specificity of incorporation. Cells grown with 1.0 MuM selenite and 8.0 mM sulfate had tRNA levels of 90 pmol Se per absorbance unit at 260 nm, or 5.4 mol Se per 100 mol tRNA. Only one selenonucleoside has been detected in enzymatic digests of E. coli tRNA. This was identified as 5-methyl-aminomethyl-2-selenouridine by comparison with synthetic standard. A new technique to separate aminoacylated from non-aminoacylated tRNA which employed affinity chromatography on immobilized anti-AMP antibodies was developed and was used to determine the amino acid acceptor specificity of the selenium-containing tRNA. At least 32% of the seleno-tRNA was specific for lysine and at least 28% was specific for glutamate. These results indicated that significant proportions of the total lysine- and glutamate accepting tRNA contained selenium. When bulk lysine accepting tRNA was purified from cells grown in 1.0 MuM selenite, about equal amounts of Se-containing and non-Se-containing lysine tRNA were obtained. The Se-containing tRNA had a distinctive UV spectrum above 300 nm due to the contribution of the 5-methylaminomethyl-2-selenouridyl residue (Lamda max 313 nm). Nuclease T-1 digestions of the Se- and non-Se-tRNAs yielded similar patterns of oligonucleotide fragments with corresponding fragments having very similar nucleotide compositions as judged by UV spectral analysis during high performance liquid chromatography. This analysis also suggested that the selenonucleoside was located in the first position of the anticodon. In this position it could influence the recognition of codons and the efficiency of protein synthesis.